PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased ß-amyloid production, altered cytokine release, and dysregulated Ca2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.

Effects of Alzheimer's Disease-Associated Risk Loci on Amyloid-ß Accumulation in the Brain of Idiopathic Normal Pressure Hydrocephalus Patients.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a dementing condition featuring characteristic symptoms, ventriculomegaly, and normal or slightly elevated cerebrospinal fluid pressure. In Alzheimer's disease (AD) patients, diffuse aggregates of amyloid-ß (Aß) and neurofibrillary hyperphosphorylated tau are detected in the neocortex of the brain, while similar accumulation of Aß is also detected in iNPH. Recent genome-wide association studies have identified several novel risk loci for AD, potentially affecting Aß-related cellular processes. Apart from the apolipoprotein E ɛ4 allele (APOE4), the risk effect of single loci is low, emphasizing the importance of the polygenic risk score approach when assessing the combined effects. OBJECTIVE: To study the effects of AD-associated individual and polygenic risk score of single nucleotide polymorphisms (SNPs) on the accumulation of Aß in the brain samples of iNPH patients. METHODS: A sample set of frontal cortex biopsies from 188 iNPH patients were divided into two groups according to the Aß pathology. After the genotyping of the AD-associated risk loci, polygenic risk score was calculated for each iNPH patient and subsequently analyzed in relation to Aß deposition. RESULTS: Apart from the APOE4, none of the SNPs revealed a statistically significant effect on the accumulation of Aß in iNPH. Also, the non-APOE4 polygenic risk score did not associate with Aß deposition. CONCLUSION: Novel AD-associated risk genes have no significant effect on Aß accumulation in the brain of iNPH patients. However, APOE4 affects the Aß deposition in the brain of iNPH and AD patients in a similar manner.

SEPT8 modulates ß-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.

Dysfunction and loss of synapses are early pathogenic events in Alzheimer's disease. A central step in the generation of toxic amyloid-ß (Aß) peptides is the cleavage of amyloid precursor protein (APP) by ß-site APP-cleaving enzyme (BACE1). Here, we have elucidated whether downregulation of septin (SEPT) protein family members, which are implicated in synaptic plasticity and vesicular trafficking, affects APP processing and Aß generation. SEPT8 was found to reduce soluble APPß and Aß levels in neuronal cells through a post-translational mechanism leading to decreased levels of BACE1 protein. In the human temporal cortex, we identified alterations in the expression of specific SEPT8 transcript variants in a manner that correlated with Alzheimer's-disease-related neurofibrillary pathology. These changes were associated with altered ß-secretase activity. We also discovered that the overexpression of a specific Alzheimer's-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aß peptides in neuronal cells. These changes were related to an increased half-life of BACE1 and the localization of BACE1 in recycling endosomes. These data suggest that SEPT8 modulates ß-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer's disease and APdE9 transgenic mouse brain and cell-based models.

Accumulation of ß-amyloid (Aß) and phosphorylated tau in the brain are central events underlying Alzheimer's disease (AD) pathogenesis. Aß is generated from amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aß pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aß40 and Aß42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aß accumulation.

Hypoxia and GABA shunt activation in the pathogenesis of Alzheimer's disease.

We have previously observed that the conversion of mild cognitive impairment to definitive Alzheimer's disease (AD) is associated with a significant increase in the serum level of 2,4-dihydroxybutyrate (2,4-DHBA). The metabolic generation of 2,4-DHBA is linked to the activation of the γ-aminobutyric acid (GABA) shunt, an alternative energy production pathway activated during cellular stress, when the function of Krebs cycle is compromised. The GABA shunt can be triggered by local hypoperfusion and subsequent hypoxia in AD brains caused by cerebral amyloid angiopathy. Succinic semialdehyde dehydrogenase (SSADH) is a key enzyme in the GABA shunt, converting succinic semialdehyde (SSA) into succinate, a Krebs cycle intermediate. A deficiency of SSADH activity stimulates the conversion of SSA into γ-hydroxybutyrate (GHB), an alternative route from the GABA shunt. GHB can exert not only acute neuroprotective activities but unfortunately also chronic detrimental effects which may lead to cognitive impairment. Subsequently, GHB can be metabolized to 2,4-DHBA and secreted from the brain. Thus, the activation of the GABA shunt and the generation of GHB and 2,4-DHBA can have an important role in the early phase of AD pathogenesis.

Genetic Variation in δ-Opioid Receptor Associates with Increased ß- and γ-Secretase Activity in the Late Stages of Alzheimer's Disease.

The agonist-induced activation of human δ-opioid receptor (δOR) has been shown to increase ß- (BACE1) and γ-secretase activities leading to increased production of amyloid-ß (Aß) peptide. We have recently shown that phenylalanine to cysteine substitution at amino acid 27 in δOR (δOR-Phe27Cys) increases amyloid-ß protein precursor processing through altered endocytic trafficking. Also, a genetic meta-analysis of the δOR-Phe27Cys variation (rs1042114) in two independent Alzheimer's disease (AD) patient cohorts indicated that the heterozygosity of δOR-Phe27Cys increases the risk of AD. Here, we investigated α-, ß-, and γ-secretase activities in human brain with respect to δOR-Phe27Cys variation in the temporal cortex of 71 subjects with varying degree of AD-related neurofibrillary pathology (Braak stages I-VI). As a result, a significant increase in ß- (p = 0.03) and γ- (p = 0.01), but not α-secretase, activities was observed in late stage AD samples (Braak stages V-VI), which were heterozygous for δOR-Phe27Cys as compared to the δOR-Phe27 and δOR-Cys27 homozygotes. The augmented ß-secretase activity was not associated with increased mRNA expression or protein levels of BACE1 in the late stage AD patients, who were heterozygous for the δOR-Phe27Cys variation. These findings suggest that δOR-Phe27Cys variation modulates ß- and γ-secretase activity in the late stages of AD likely via post-translational mechanisms other than alterations in the mRNA or protein levels of BACE1, or, in the expression of γ-secretase complex components.

Transcriptomics and mechanistic elucidation of Alzheimer's disease risk genes in the brain and in vitro models.

In this study, we have assessed the expression and splicing status of genes involved in the pathogenesis or affecting the risk of Alzheimer's disease (AD) in the postmortem inferior temporal cortex samples obtained from 60 subjects with varying degree of AD-related neurofibrillary pathology. These subjects were grouped based on neurofibrillary pathology into 3 groups: Braak stages 0-II, Braak stages III-IV, and Braak stages V-VI. We also examined the right frontal cortical biopsies obtained during life from 22 patients with idiopathic shunt-responding normal pressure hydrocephalus, a disease that displays similar pathologic alterations as seen in AD. These 22 patients were categorized according to dichotomized amyloid-ß positive or negative pathology in the biopsies. We observed that the expression of FRMD4A significantly decreased, and the expression of MS4A6A significantly increased in relation to increasing AD-related neurofibrillary pathology. Moreover, the expression of 2 exons in both CLU and TREM2 significantly increased with increase in AD-related neurofibrillary pathology. However, a similar trend toward increased expression in CLU and TREM2 was observed with most of the studied exons, suggesting a global change in the expression rather than altered splicing. Correlation of gene expression with well-established AD-related factors, such as α-, ß-, and γ-secretase activities, brain amyloid-ß42 levels, and cerebrospinal fluid biomarkers, revealed a positive correlation between ß-secretase activity and the expression of TREM2 and BIN1. In expression quantitative trait loci analysis, we did not detect significant effects of the risk alleles on gene expression or splicing. Analysis of the normal pressure hydrocephalus biopsies revealed no differences in the expression or splicing profiles of the studied genes between amyloid-ß positive and negative patients. Using the protein-protein interaction-based in vitro pathway analysis tools, we found that downregulation of FRMD4A associated with increased APP-ß-secretase interaction, increased amyloid-ß40 secretion, and altered phosphorylation of tau. Taken together, our results suggest that the expression of FRMD4A, MS4A6A, CLU, and TREM2 is altered in relation to increasing AD-related neurofibrillary pathology, and that FRMD4A may play a role in amyloidogenic and tau-related pathways in AD. Therefore, investigation of gene expression changes in the brain and effects of the identified genes on disease-associated pathways in vitro may provide mechanistic insights on how alterations in these genes may contribute to AD pathogenesis.

Effects of Alzheimer's disease-associated risk loci on cerebrospinal fluid biomarkers and disease progression: a polygenic risk score approach.

BACKGROUND: Several risk loci for Alzheimer's disease (AD) have been identified during recent years in large-scale genome-wide association studies. However, little is known about the mechanisms by which these loci influence AD pathogenesis. OBJECTIVE: To investigate the individual and combined risk effects of the newly identified AD loci. METHODS: Association of 12 AD risk loci with AD and AD-related cerebrospinal fluid (CSF) biomarkers was assessed. Furthermore, a polygenic risk score combining the effect sizes of the top 22 risk loci in AD was calculated for each individual among the clinical and neuropathological cohorts. Effects of individual risk loci and polygenic risk scores were assessed in relation to CSF biomarker levels as well as neurofibrillary pathology and different biochemical measures related to AD pathogenesis obtained from the temporal cortex. RESULTS: Polygenic risk scores associated with CSF amyloid-ß42 (Aß42) levels in the clinical cohort, and with soluble Aß42 levels and γ-secretase activity in the neuropathological cohort. The γ-secretase effect was independent of APOE. APOE-ε4 associated with CSF Aß42 (p < 0.001) levels. For the other risk loci, no significant associations with AD risk or CSF biomarkers were detected after multiple testing correction. CONCLUSIONS: AD risk loci polygenically contribute to Aß pathology in the CSF and temporal cortex, and this effect is potentially associated with increased γ-secretase activity.

Increased γ-secretase activity in idiopathic normal pressure hydrocephalus patients with ß-amyloid pathology.

The potential similarity between the brain pathology of idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer disease (AD) is intriguing and thus further studies focusing on the underlying molecular mechanisms may offer valuable information for differential diagnostics and the development of treatments for iNPH. Here, we investigated ß- and γ-secretase activities in relation to amyloid-ß (Aß) pathology in the brain tissue samples collected from iNPH and AD patients. ß- and γ-secretase activities were measured from the frontal cortical biopsies of 26 patients with suspected iNPH as well as post-mortem tissue samples from the inferior temporal cortex of 74 AD patients and eight subjects without neurofibrillary pathology. In iNPH samples with detectable Aß plaques, γ-secretase activity was significantly increased (∼ 1.6-fold) when compared to iNPH samples without Aß plaques (p = 0.009). In the AD samples, statistically significant differences in the γ-secretase activity were not observed with respect to disease severity (mild, moderate and severe AD according to neurofibrillary pathology). Conversely, ß-secretase activity was unaltered in iNPH samples with or without Aß plaques, while it was significantly increased in relation to disease severity in the AD patients. These results show for the first time increased γ-secretase but not ß-secretase activity in the biopsy samples from the frontal cortex of iNPH patients with AD-like Aß pathology. Conversely, the opposite was observed in these secretase activities in AD patients with respect to neurofibrillary pathology. Despite the resemblances in the Aß pathology, iNPH and AD patients appear to have marked differences in the cellular mechanisms responsible for the production of Aß.

Effects of NR1H3 genetic variation on the expression of liver X receptor α and the progression of Alzheimer's disease.

Alzheimer's disease (AD) has been postulated to involve defects in the clearance of amyloid-ß (Aß). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aß. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aß42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aß42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aß42.

Elucidation of the BACE1 regulating factor GGA3 in Alzheimer's disease.

Golgi-localized γ-ear-containing ADP-ribosylation factor-binding protein (GGA3) is a central regulator of trafficking and degradation of BACE1 (ß-site AßPP-cleaving enzyme), the rate-limiting enzyme in the production of amyloid-ß (Aß) in Alzheimer's disease (AD). Here, we assessed the potential role of GGA3 in AD pathogenesis using independent neuropathological, case-control, and family-based human sample cohorts. Increased BACE1 levels coincided with decreased GGA3 levels and with elevated phosphorylation status of eIF2α-Ser51 in the temporal cortex of AD patients as compared to age-matched controls. Severity of the disease did not alter mRNA or protein levels of GGA3 in the inferior temporal cortex of AD patients, while a positive correlation between GGA3 and the levels of total, but not phosphorylated, tau was observed. Genetically, we did not observe consistent evidence for association between AD risk and common GGA3 polymorphisms across a number of independent sample cohorts. However, a nominally significant association was observed with rs2242230 (p < 0.05) among the Finnish case-control cohort. Accordingly, mRNA and protein levels of GGA3 in the inferior temporal cortex of AD patients did not significantly correlate with rs2242230 genotype status. While the present study indicates that GGA3 is involved in the cellular processes relevant for AD pathogenesis, the genetic data do not support the idea that common GGA3 polymorphisms would contribute to AD risk.

Bepridil decreases Aß and calcium levels in the thalamus after middle cerebral artery occlusion in rats.

Alzheimer's disease (AD) and cerebral ischaemia share similar features in terms of altered amyloid precursor protein (APP) processing and ß-amyloid (Aß) accumulation. We have previously shown that Aß and calcium deposition, and ß-secretase activity, are robustly increased in the ipsilateral thalamus after transient middle cerebral artery occlusion (MCAO) in rats. Here, we investigated whether the non-selective calcium channel blocker bepridil, which also inhibits ß-secretase cleavage of APP, affects thalamic accumulation of Aß and calcium and in turn influences functional recovery in rats subjected to MCAO. A 27-day bepridil treatment (50 mg/kg, p.o.) initiated 2 days after MCAO significantly decreased the levels of soluble Aß40, Aß42 and calcium in the ipsilateral thalamus, as compared with vehicle-treated MCAO rats. Expression of seladin-1/DHCR24 protein, which is a potential protective factor against neuronal damage, was decreased at both mRNA and protein levels in the ipsilateral thalamus of MCAO rats. Conversely, bepridil treatment restored seladin-1/DHCR24 expression in the ipsilateral thalamus. Bepridil treatment did not significantly affect heme oxygenase-1- or NAD(P)H quinone oxidoreductase-1-mediated oxidative stress or inflammatory responses in the ipsilateral thalamus of MCAO rats. Finally, bepridil treatment mitigated MCAO-induced alterations in APP processing in the ipsilateral thalamus and improved contralateral forelimb use in MCAO rats. These findings suggest that bepridil is a plausible therapeutic candidate in AD or stroke owing to its multifunctional role in key cellular events that are relevant for the pathogenesis of these diseases.

Genetic analysis of genes involved in amyloid-ß degradation and clearance in Alzheimer's disease.

Accumulation of amyloid ß-peptide (Aß) in the brain of Alzheimer's disease (AD) patients has been postulated to reflect defects in Aß degradation or clearance. Here, we selected 12 genes (MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9) involved in Aß catabolism on the basis of PubMed-based literature search and elucidated their genetic role in AD among Finnish case-control cohort consisting of total ∼1,300 AD patients and control subjects. Thirty one single nucleotide polymorphisms (SNPs) were selected for genotyping. In a smaller subset of AD patients, cerebrospinal fluid (CSF) levels of Aß42 (n = 124), total-tau (n = 59), and phospho-tau (n = 54) analyses were performed with respect to SNPs. Moreover, age of onset analyses with respect to the studied SNPs were conducted among the AD patient cohort (n = 642). Association analysis of the liver X receptor α (NR1H3) gene SNPs showed a protective effect for C allele carriers of rs7120118 (OR = 0.70, 95% CI 0.53-0.93), while the total-tau and phospho-tau levels in CSF were decreased in AD patients carrying the C allele. Also, a decrease in the age of onset was observed in AD patients carrying the A allele of rs723744 and the C allele of rs3794884 in transthyretin (TTR) gene. However, after adjusting the p-values for multiple comparisons, these results were not statistically significant, suggesting that genetic variations in MMEL1, ECE1, ECE2, AGER, PLG, PLAT, NR1H3, MMP3, LRP1, TTR, NR1H2, and MMP9 genes do not play major role among the Finnish AD patient cohort.

Effects of memantine and donepezil on cortical and hippocampal acetylcholine levels and object recognition memory in rats.

This preclinical study investigated the ability of memantine (MEM) to stimulate brain acetylcholine (ACh) release, potentially acting synergistically with donepezil (DON, an acetylcholinesterase inhibitor). Acute systemic administration of either MEM or DON to anesthetized rats caused dose-dependent increases of ACh levels in neocortex and hippocampus, and the combination of MEM (5 mg/kg) and DON (0.5 mg/kg) produced significantly greater increases than either drug alone. To determine whether ACh release correlated with cognitive improvement, rats with partial fimbria-fornix (FF) lesions were treated with acute or chronic MEM or DON. Acute MEM treatment significantly elevated baseline hippocampal ACh release but did not significantly improve task performance on a delayed non-match-to-sample (DNMS) task, whereas chronic MEM treatment significantly improved DNMS performance but only marginally elevated baseline ACh levels. Acute or chronic treatment with DON (in the presence of neostigmine to allow ACh collection) did not significantly improve DNMS performance or alter ACh release. In order to investigate the effect of adding MEM to ongoing DON therapy, lesioned rats pretreated with DON for 3 weeks were given a single intraperitoneal dose of MEM. MEM significantly elevated baseline hippocampal ACh levels, but did not significantly improve DNMS task scores compared to chronic DON-treated animals. These data indicate that MEM, in addition to acting as an NMDA receptor antagonist, can also augment ACh release; however, in this preclinical model, increased ACh levels did not directly correlate with improved cognitive performance.

Cysteine 27 variant of the delta-opioid receptor affects amyloid precursor protein processing through altered endocytic trafficking.

Agonist-induced activation of the δ-opioid receptor (δOR) was recently shown to augment ß- and γ-secretase activities, which increased the production of ß-amyloid peptide (Aß), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the δOR variant with a phenylalanine at position 27 (δOR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (δOR-Cys27) variant. For this study, we expressed these variants in human SH-SY5Y and HEK293 cells expressing exogenous or endogenous amyloid precursor protein (APP) and assessed the effects on APP processing. Expression of δOR-Cys27, but not δOR-Phe27, resulted in a robust accumulation of the APP C83 C-terminal fragment and the APP intracellular domain, while the total soluble APP and, particularly, the ß-amyloid 40 levels were decreased. These changes upon δOR-Cys27 expression coincided with decreased localization of APP C-terminal fragments in late endosomes and lysosomes. Importantly, a long-term treatment with a subset of δOR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the δOR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the δOR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.

An association study of 21 potential Alzheimer's disease risk genes in a Finnish population.

Alzheimer's disease (AD) is a genetically complex disorder encompassing several individual susceptibility genes with low risk effects. To assess the risk gene effects in a cohort consisting of ∼ 1300 Finnish AD patients and controls, 21 candidate gene polymorphisms were selected for genotyping on the basis of the meta-analyses retrieved from the AlzGene database. A significant genotype and allele association with AD was observed with rs1800629 in the tumor necrosis factor α (TNF). Risk analysis revealed a protective effect for the minor allele carriers of rs1800629. This suggests that genetic alteration in TNF gene may play a role in AD.

A novel delayed non-match to sample object recognition task that allows simultaneous in vivo microdialysis.

We present a modification of the widely used delayed non-match to sample (DNMS) paradigm for assessment of object recognition memory that can be combined with simultaneous in vivo microdialysis. The present study provides evidence that hippocampal ACh release increases from baseline during active exploration of the test environment and an empty test board, but a specific further increase is seen during the recognition memory task performance. This novel experimental model offers a good tool to study the impact of selective lesions or pharmacological manipulation simultaneously on neurotransmitter levels and memory task performance.

CALHM1 P86L polymorphism does not alter amyloid-beta or tau in cerebrospinal fluid.

Recently, the P86L alteration in CALHM1 (calcium homeostasis modulator-1) was reported to be associated with Alzheimer's disease (AD). Moreover, the risk allele increased amyloid-beta (A beta) levels in conditioned media from cultured cells. Therefore, we hypothesized that CALHM1 P86L may modulate A beta or tau levels in cerebrospinal fluid (CSF). Nearly 200 individuals with AD or other cognitive disorders were included for CSF analysis and CALHM1 genotyping. No significant differences in CSF levels of A beta 42, tau or phospho-tau were found across the various CALHM1 genotypes. In conclusion, we found no evidence that CALHM1 P86L is associated with altered CSF levels of the investigated AD biomarkers.

Down-regulation of seladin-1 increases BACE1 levels and activity through enhanced GGA3 depletion during apoptosis.

Seladin-1 is a neuroprotective protein selectively down-regulated in brain regions affected in Alzheimer disease (AD). Seladin-1 protects cells against beta-amyloid (Abeta) peptide 42- and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the beta-secretase (BACE1) function and beta-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that approximately 60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or Abeta secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized gamma-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced beta-amyloidogenic processing of APP and ultimately increased Abeta production. These adverse changes associated with decreased cell viability in seladin-1 siRNA-transfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased beta-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis.

Quantitative determination of acetylcholine in microdialysis samples using liquid chromatography/atmospheric pressure spray ionization mass spectrometry.

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples.